Isolation of Neurospora mitochondrial tRNA


This chapter discusses the isolation of Neurospora mitochondrial tRNA. Large quantities of highly purified mitochondria are obtained from Neurospora crassa with the use of zonal rotors. The ability to obtain these organelles in high yields has been instrumental in demonstrating the existence of unique species of tRNAs and aminoacyl-tRNA synthetases in mitochondria and, more recently, to isolate and purify initiator methionine tRNA from these organelles. Neither methionine nor phenylalanine tRNAs from mitochondria are detected in total cell preparations from N. crassa. This situation is probably due to the presence of relatively small amounts of mitochondrial tRNAs in Neurospora and/or the lability of these molecules because of their high (A+U) contents. Thus, in order to detect and purify mitochondrial tRNAs, large amounts of N. crassa mitochondria is isolated free of cytoplasmic contamination. The methods of mitochondrial isolation employed are derived from those of Hall and Greenawalt and in some respects are similar to the small-scale isolation of mitochondria. © 1979, Elsevier Inc. All rights reserved.

Publication Title

Methods in Enzymology